The detection of cytokine release by ELISPOT assay and the quantification and identification of intracellular cytokines by flow cytometry are both widely used as indices of cell mediated immune responses. In the so-called ELISPOT assay, cytokines released from immobilised cells typically interact with an immunoassay, such as an ELISA assay, to produce a coloured “spot” on the assay plate providing both qualitative (e.g. type of immune protein) and quantitative (number or proportion of cells responding) information. The standardisation of these assays requires the development of reliable reference standards for monitoring intracellular cytokine levels and cytokine release in test samples, thus allowing comparability between different laboratories and assays.
Stimulated (cytokine positive), fixed, cryopreserved cells are available commercially for intracellular flow cytometry (Becton Dickinson, Oxford, UK), and freeze dried unstimulated cells are available either as controls for surface staining or for purposes of haematology analysis (Beckman Coulter UK Ltd, High Wycombe, UK).
For ELISPOT assays, or other assays where cytokine release from cells is monitored, only cryopreserved live cells from individual donors are currently available.
The presently-available approaches to the provision of a standardised reference material have a number of drawbacks: In order to standardise reference materials over a large number of laboratories world-wide, and to provide reference materials that may be used over many years, a large amount of stable material is required. However, live cells from multiple donors cannot be pooled to make large single batches due to mixed lymphocyte reactions, resulting in cell death and over-expression of cytokines.
Cryopreserved cells also require specialised storage using e.g. liquid nitrogen and shipment on dry ice, thus increasing costs. There is also the risk of thawing and refreezing during shipment, in the event of power failure to refrigeration devices, say, leading to deterioration of the material, so rendering it useless as a reference standard. Furthermore, cyropreserved cells must be carefully thawed to ensure consistency of responses. It has proven difficult to obtain consistent results between laboratories using this approach.
Measurement of cell proliferation in mammalian cells is also an important technique for e.g. the assessment of the effects of exogenous agents on a cell's ability or propensity to divide. For example, such assays may be used for detecting the marked proliferation of cells of the immune system following an immune response. A number of techniques are currently used wherein cells are labeled with a detectable marker that is shared between daughter cells following cell division.
One such technique uses the fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE or CFDA, SE). The technique is described by Lyons, B. in Immunol. Cell Biol 1999; 77(6):509-515 in which the author reports that “The technique can be used both in vitro and in vivo, allowing eight to 10 successive divisions to be resolved by flow cytometry. Furthermore, viable cells from defined generation numbers can be sorted by flow cytometry for functional analysis”. Other techniques include the incorporation of bromodeoxyuridine or tritiated thymidine into the cells.
Whilst these assays are well known, a difficulty that arises is the provision of reference standards for cross-calibration and quality control when the assays are used across different analysis laboratories, or are used successively over long periods of time, e.g. during clinical longitudinal studies.
The lack of suitable reference materials for such cytokine and cell proliferation assays hinders robust testing methodologies, especially in respect of comparability between different laboratories and assays. It is amongst the objects of the present invention to attempt a solution to these problems.